U.S. patent application number 15/730382 was filed with the patent office on 2018-04-19 for n-acetyl-alpha-d-glucosaminidase deficiency compositions and methods.
The applicant listed for this patent is ALEXION PHARMACEUTICALS, INC.. Invention is credited to Christen D. Forbes, Andrew Hutchinson, Nina Jain, Rui-Ru Ji.
Application Number | 20180105879 15/730382 |
Document ID | / |
Family ID | 61904311 |
Filed Date | 2018-04-19 |
United States Patent
Application |
20180105879 |
Kind Code |
A1 |
Ji; Rui-Ru ; et al. |
April 19, 2018 |
N-ACETYL-alpha-D-GLUCOSAMINIDASE DEFICIENCY COMPOSITIONS AND
METHODS
Abstract
Compositions and methods relating to potentially pathogenic
mutations in the nucleotide sequence of a human NAGLU gene. Some
NAGLU gene variants have been discovered to be associated with
reduced N-acetyl-.alpha.-D-glucosaminidase (NAGLU) activity.
Inventors: |
Ji; Rui-Ru; (Belmont,
MA) ; Hutchinson; Andrew; (Stamford, CT) ;
Jain; Nina; (North Andover, MA) ; Forbes; Christen
D.; (North Haven, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALEXION PHARMACEUTICALS, INC. |
New Haven |
CT |
US |
|
|
Family ID: |
61904311 |
Appl. No.: |
15/730382 |
Filed: |
October 11, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62408281 |
Oct 14, 2016 |
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62455905 |
Feb 7, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/156 20130101; C12Y 302/0105 20130101; A61K 38/47
20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; A61K 38/47 20060101 A61K038/47 |
Claims
1. A method for detecting a potentially pathogenic mutation in a
human NAGLU nucleotide sequence, the method comprising: (a)
providing a biological sample comprising a nucleic acid; and (b)
performing a genetic analysis on the biological sample to detect
the presence of a potentially pathogenic mutation in a human NAGLU
nucleotide sequence, wherein the potentially pathogenic mutation
comprises at least one of (i) a nucleotide sequence mutation
encoding an amino acid substitution at an amino acid position
selected from the group consisting of L35, M338, R541, and L622
relative to the wild-type human NAGLU amino acid sequence (SEQ ID
NO:1); or (ii) a nucleotide sequence mutation selected from the
group consisting of c.104T>C, c.1012A>G, c.1621C>T,
c.1865T>C, c.1128_1138del11, c.351delG, IVS2+5G>A, and
c.1700_1708del9 relative to the wild-type human NAGLU cDNA sequence
(SEQ ID NO:2).
2. The method of claim 1, wherein the mutation comprises an
N-acetyl-.alpha.-D-glucosaminidase (NAGLU) activity-reducing
mutation.
3. The method of claim 1, wherein the mutation occurs in
combination with a second mutation in the human NAGLU nucleotide
sequence.
4. The method of claim 1, wherein the potentially pathogenic
mutation does not cause a frame shift.
5. The method of claim 1, wherein the potentially pathogenic
mutation causes a frame shift.
6. The method of claim 1, wherein performing a genetic analysis on
the biological sample comprises performing whole transcriptome
sequencing, whole exome sequencing, whole genome sequencing, or
hybridization to a DNA microarray.
7. The method of claim 1, wherein the biological sample is obtained
from a patient, and wherein the method further comprises diagnosing
the patient with Mucopolysaccharidosis IIIB (MPS IIIB) when the
presence of a potentially pathogenic mutation in the NAGLU
nucleotide sequence of the patient is detected.
8. The method of claim 7, further comprising administering a
therapeutically effective amount of a recombinant human NAGLU to
the patient.
9.-15. (canceled)
16. The method of claim 7, wherein the patient is a pediatric
patient.
17. The method of claim 7, wherein the patient is an adult
patient.
18. The method of claim 7, wherein diagnosing the patient with MPS
IIIB comprises determining that the patient has biallelic
pathogenic variants in the NAGLU gene.
19.-23. (canceled)
24. The method of claim 8, wherein the patient is a pediatric
patient.
25. The method of claim 8, wherein the patient is an adult
patient.
26. The method of claim 1, wherein the biological sample is
obtained from a patient, and wherein the method further comprises
screening for the presence of MPS IIIB in a patient, the method
comprising: determining the nucleotide sequence of one or more of
the patient's NAGLU alleles; and determining whether one or more of
the patient's NAGLU alleles comprises the potentially pathogenic
mutation.
27. The method of claim 26, further comprising determining whether
the patient has biallelic pathogenic variants in the NAGLU
gene.
28. The method of claim 26, further comprising screening for the
presence of additional disease in the patient.
29. The method of claim 28, wherein the additional disease
comprises a Mendelian disorder.
30. A kit for detecting a potentially pathogenic mutation in a
human NAGLU nucleotide sequence, said kit comprising at least one
oligonucleotide primer or allele-specific probe specific for a
potentially pathogenic mutation comprising at least one of (i) a
nucleotide sequence mutation encoding an amino acid substitution at
an amino acid position selected from the group consisting of L35,
M338, R541, and L622 relative to the wild-type human NAGLU amino
acid sequence (SEQ ID NO:1); or (ii) a nucleotide sequence mutation
selected from the group consisting of c.104T>C, c.1012A>G,
c.1621C>T, c.1865T>C, c.1128_1138del11, c.351delG,
IVS2+5G>A, and c.1700_1708del9 relative to the wild-type human
NAGLU cDNA sequence (SEQ ID NO:2); and instructions relating to
detecting mutations in the NAGLU nucleotide sequence.
31. (canceled)
32. (canceled)
33. A vector operably encoding a polynucleotide comprising a
mutated human NAGLU nucleotide sequence mutation selected from the
group consisting of c.104T>C, c.1012A>G, c.1621C>T,
c.1865T>C, c.1128_1138del11, c.351delG, IVS2+5G>A, and
c.1700_1708del9 relative to the wild-type human NAGLU cDNA sequence
(SEQ ID NO:2).
34. A host cell comprising the vector of claim 33.
35.-38. (canceled)
Description
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 62/408,281 filed Oct. 14, 2016, and
62/455,905, filed Feb. 7, 2017, each of which is incorporated
herein by reference in its entirety.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing electronically
submitted via EFS-Web to the United States Patent and Trademark
Office as an ASCII text file entitled "535-0003_ST25.txt" having a
size of 10 kilobytes and created on Sep. 25, 2017. The information
contained in the Sequence Listing is incorporated by reference
herein.
BACKGROUND
[0003] Mucopolysaccharidosis IIIB (MPS IIIB, also known Sanfilippo
Syndrome B, N-acetyl-.alpha.-D-glucosaminidase deficiency, and
NAGLU deficiency) is a genetic, progressive, systematic, rare and
devastating autosomal recessive lysosomal storage disease (LSD)
caused by a deficiency in N-acetyl-.alpha.-D-glucosaminidase (i.e.,
N-acetyl-alpha-D-glucosaminidase abbreviated as NAGLU). NAGLU is a
lysosomal enzyme required for the degradation of heparan sulfate as
part of the stepwise breakdown of glycosaminoglycans (GAG) in the
lysosome. In patients with MPS IIIB, genetic mutations result in a
marked decrease in NAGLU enzyme activity, which leads to the
accumulation of heparan sulfate (HS) in the brain and other organs.
The accumulation of HS leads to progressive brain atrophy,
neurocognitive decline, behavioral disturbances, speech loss,
increasing loss of mobility, and premature death. With over almost
200 different mutations identified to date MPS IIIB exhibits
extensive molecular and genetic heterogeneity.
[0004] Approximately 1 out of 200,000 births is affected by MPS
IIIB, and the deficiency mainly manifests in young children. MPS
IIIB typically presents during the first few years of life, and
patients have a greater than 50 percent mortality rate by 17 years
of age. After initial symptom-free interval, patients suffering
from MPS IIIB usually present with a slowing of mental development
and behavioral problems, followed by progressive intellectual
decline resulting in severe mental retardation, dementia and motor
disease. Acquisition of speech is slow and incomplete. Profoundly
affected patients may present delayed psychomotor and speech
development as early as 2 years of age. The disease usually
progresses to increasing behavioral disturbance and sleep
disturbance. Although the clinical features are mainly
neurological, patients often develop diarrhea, carious teeth, an
enlarged liver and spleen, stiff joints, hirsteness and/or coarse
hair and may exhibit blood-clotting problems. In the final stage of
the illness, patients become immobile and unresponsive and develop
swallowing difficulties and seizure. The life-span of an affected
child typically does not extend beyond late teens to early
twenties.
[0005] There are no approved treatments for patients with MPS IIIB.
Current supportive care is palliative for behavioral problems,
sleep disturbances, seizures, and other complications, and does not
address the root cause of MPS IIIB or stop disease progression.
However, investigational enzyme replacement therapy utilizing a
recombinant human NAGLU (rhNAGLU) is currently being investigated
in clinical trials for patients with MPS IIIB. The enzyme is a
recombinant form of the N-acetyl-.alpha.-D-glucosaminidase (NAGLU)
enzyme intended to replace the missing or deficient NAGLU enzyme,
with the goal of to reduce accumulated HS. See WO2013/055888,
published Apr. 18, 2013; US Pat. Appl. Publication 2013/0095092
(Quinn et al.), published Apr. 18, 2013. A recombinant human NAGLU
preferably omits all or a portion of the signal peptide (amino
acids 1-13) that is present in naturally occurring human NAGLU (SEQ
ID NO:1).
SUMMARY OF THE INVENTION
[0006] In one aspect, the invention describes mutations in the
nucleotide sequence of a human NAGLU gene that have been discovered
to be associated with reduced N-acetyl-.alpha.-D-glucosaminidase
(NAGLU) activity. These mutations may be referred to herein as
"NAGLU activity-reducing mutations." In some embodiments, the
activity of the NAGLU encoded by the mutated gene is reduced so far
that it is no longer detectable or is eliminated. In other
embodiments, the activity of the NAGLU encoded by the mutated gene
is reduced but still detectable. In some embodiments, the NAGLU
activity-reducing mutation is present in the coding region of a
human NAGLU gene. A human NAGLU gene that is mutated such that it
produces an NAGLU with reduced activity is suspected of being
pathogenic and as such, is referred to herein as a potentially
pathogenic NAGLU gene variant or allelic variant. In some
embodiments, the mutation results in an amino acid substitution or
deletion in the NAGLU encoded by the mutated NAGLU gene. In some
embodiments, the mutation causes a truncation of NAGLU. The
mutation can be, for example, a missense mutation or a nonsense
mutation. In some embodiments, the mutation causes misregulation of
precursor RNA splicing, and/or alternative precursor mRNA splicing.
In some embodiments, the mutation can be an insertion or deletion
of one or more amino acids, and optionally can cause a frameshift
in the NAGLU encoded by the mutated NAGLU gene.
[0007] One or more of the NAGLU activity-reducing mutations
described herein may occur alone, or in combination with each other
and/or in combination with one or more known mutations in the human
NAGLU gene.
[0008] In another aspect, the invention describes seven additional
novel clinically identified mutations in the nucleotide sequence of
a human NAGLU gene. A human NAGLU gene that includes one or more of
these seven novel mutations is suspected of being pathogenic and as
such, is also referred to herein as a potentially pathogenic NAGLU
gene variant.
[0009] In another aspect, the invention provides a method for
detecting the presence or absence of an NAGLU activity-reducing
mutation in the nucleotide sequence of a NAGLU gene of a human
subject. This method makes possible a determination as to whether
the subject possesses a potentially pathogenic NAGLU gene variant,
also referred to as a potentially pathogenic NAGLU allele or
allelic variant. The presence of a potentially pathogenic gene
variant may be indicative of MPS IIIB. Any convenient detection
method can be used to detect an NAGLU activity-reducing
mutation.
[0010] In another aspect, the invention provides a method for
detecting the presence or absence of a novel clinically identified
mutation, as described herein, in the nucleotide sequence of a
NAGLU gene of a human subject. This method makes possible a
determination as to whether the subject possesses a potentially
pathogenic NAGLU gene variant, also referred to as a potentially
pathogenic NAGLU allele or allelic variant. The presence of a
potentially pathogenic gene variant may be indicative of MPS IIIB.
Any convenient detection method can be used to detect a novel
clinically identified mutation.
[0011] In another aspect, the invention provides a method for
diagnosing Mucopolysaccharidosis IIIB (MPS IIIB, also known as
Sanfilippo Syndrome B) in a human subject. The nucleotide sequence
of the NAGLU gene of the subject, or related mRNA or cDNA, is
analyzed to determine the presence of a mutation associated with
reduced NAGLU activity, wherein reduced NAGLU activity of the gene
variant is indicative MPS IIIB. Additionally or alternatively, the
nucleotide sequence is analyzed to determine the presence of a
novel clinically identified mutation, as described herein. Mutation
detection can be used alone in combination with other diagnostic
factors. The diagnostic method of the invention optionally includes
treating the patient for MPS IIIB.
[0012] In some embodiments of the detection or diagnostic method,
the NAGLU gene is present in or isolated from a biological sample
obtained from a human subject. The subject can be a child or an
adult. The polynucleotide evaluated for presence or absence of the
mutation can be, for example, DNA, RNA or cDNA. In some embodiments
of the detection or diagnostic method, genetic analysis for the
presence or absence of a mutation takes the form of exomic or
genomic analysis performed on nucleic acids obtained from the
subject.
[0013] In another aspect, the invention provides a kit for
detecting an NAGLU activity-reducing mutation and/or a novel
clinically identified mutation in the nucleotide sequence of a
NAGLU gene. In some embodiments, the kit includes at least one
oligonucleotide primer specific for an NAGLU activity-reducing
NAGLU gene mutation or a novel clinically identified NAGLU gene
mutation as described herein, and instructions relating to
detecting mutations in the nucleotide sequence of a NAGLU gene. In
some embodiments, the kit includes at least one allele-specific
oligonucleotide probe for an NAGLU activity-reducing NAGLU gene
mutation or the novel clinically identified NAGLU gene mutation as
described herein and instructions relating to detecting mutations
in the nucleotide sequence of a NAGLU gene. Optionally the kit
includes a multiplicity of primers or probes to permit the
detection of a multiplicity of mutations in the nucleotide sequence
of a human NAGLU gene, thereby increasing the diagnostic or
screening efficiency of the kit.
[0014] In another aspect, the invention provides a method for
treating a patient afflicted with, or suspected of being afflicted
with MPS IIIB, wherein the nucleotide sequence of a NAGLU gene of
the patient contains an NAGLU activity-reducing mutation and/or a
novel clinically identified mutation as described herein. In some
embodiments, the patient is treated with enzyme replacement
therapy, for example by using recombinant NAGLU. In some
embodiments, the patient is treated with therapeutic
polynucleotides. Exemplary treatments and therapeutic agents for
use with MPS IIIB patients are described, without limitation, in
WO2013/055888, published Apr. 18, 2013; US Pat. Appl. Publication
2013/0095092 (Quinn et al.), published Apr. 18, 2013; and
WO/2016/054025, published Jul. 4, 2015.
Definitions
[0015] For convenience, certain terms employed in the
specification, examples, and appended claims are set forth herein
to illustrate and define the meaning and scope of the various terms
used to describe the present invention.
"NAGLU" as used herein refers to
"N-acetyl-.alpha.-D-glucosaminidase," and the two terms are used
interchangeably throughout the specification. The NAGLU can be a
human protein, i.e., human N-acetyl-.alpha.-D-glucosaminidase.
[0016] NAGLU is encoded by the NAGLU gene, which is present in the
human population in a variety of allelic forms. The most prevalent
allele, referred to herein as "wild-type" (WT), encodes a
functional ("wild-type") NAGLU. Wild-type NAGLU is referred to
herein as having "normal" activity. The amino acid sequence of a
wild-type NAGLU (SEQ ID NO:1) is shown in FIG. 1, and the coding
sequence of a wild-type human NAGLU gene (SEQ ID NO:2) is shown in
FIG. 2. The x-ray crystallographic structure of human NAGLU protein
is reported in U.S. Pat. No. 8,775,146. NAGLU catalyzes the
hydrolysis of terminal non-reducing N-acetyl-D-glucosamine residues
in N-acetyl-.alpha.-D-glucosaminides, which is referred to herein
as "NAGLU activity." NAGLU activity is also known as NAG activity,
.alpha.-acetylglucosaminidase activity,
N-acetyl-.alpha.-glucosaminidase activity,
.alpha.-N-acetylglucosaminidase activity, and
N-acetyl-.alpha.-D-glucosaminidase activity according to EMBL-EBI
(2016). "NAGLU activity" can be measured in vitro, for example, by
the cleavage of the fluorogenic substrate, 4-methylumbelliferyl
N-acetyl-.alpha.-D-glucosaminide (4MU-NAG). Cleavage of 4MU-NAG can
be detected, for example, by excitation at about 360 nm and
emission at 460 nm of the released fluorophore,
4-methylumbelliferone (4-MU). Results can be reported in relative
fluorescence units (RFU). For example, the amount of substrate
cleaved in a 30 minute endpoint assay can be quantified relative to
a 4-MU standard curve, and one unit (U) of activity can be defined
as the amount of enzyme required to cleave 1 micromole of 4MU-NAG
per minute at 37.degree. C. Accordingly, functional fragments or
variants of NAGLU include fragments or variants that have NAGLU
activity, e.g., the ability to hydrolyze terminal non-reducing
N-acetyl-D-glucosamine residues in
N-acetyl-.alpha.-D-glucosaminides.
[0017] As used herein "exogenous NAGLU" refers to NAGLU that is not
naturally produced by a patient. For example, exogenous NAGLU
includes recombinant NAGLU protein that is administered to a
patient, NAGLU protein that is isolated from a person or animal and
administered to a patient, and NAGLU protein that is produced
(i.e., expressed) in a patient as a result of administration of
NAGLU-encoding RNA and/or DNA or another treatment that increases
expression of endogenous NAGLU protein.
[0018] As used herein an "NAGLU associated disease" is a disease or
condition which is mediated by NAGLU activity or is associated with
aberrant NAGLU expression or activity. An example of an NAGLU
associated disease includes, but is not limited to, NAGLU
deficiency such as Mucopolysaccharidosis type IIIB (also known as
Sanflippo Syndrome B).
[0019] "Intravenous injection," often medically referred to as IV
push or bolus injection, refers to a route of administration in
which a syringe is connected to the IV access device and the
medication is injected directly, typically rapidly and occasionally
up to a period of 15 minutes if it might cause irritation of the
vein or a too-rapid effect. Once a medicine has been injected into
the fluid stream of the IV tubing, there must be some means of
ensuring that it gets from the tubing to the patient. Usually this
is accomplished by allowing the fluid stream to flow normally and
thereby carry the medicine into the bloodstream. However, in some
cases a second fluid injection, sometimes called a "flush," is used
following the first injection to facilitate the entering of the
medicine into the bloodstream.
[0020] "Intravenous infusion" refers to a route of administration
in which medication is delivered over an extended period of time.
For example, the medication can be delivered to a patient over a
period of time between 1 and 8 hours. The medication can also be
delivered to a patient over a period of about 1, about 2, about 3,
about 4, about 5, about 6, about 7, or about 8 hours. To accomplish
an intravenous infusion, an IV gravity drip or an IV pump can be
used. IV infusion is typically used when a patient requires
medications only at certain times and does not require additional
intravenous fluids (e.g., water solutions which can contain sodium,
chloride, glucose, or any combination thereof) such as those that
restore electrolytes, blood sugar, and water loss.
[0021] The term "patient" as used herein refers to any person
receiving or who has received or is to receive medical care or
treatment, e.g., as directed by a medical care provider.
[0022] A "therapeutically effective" amount or a "therapeutically
effective" dose, as the terms are used herein, refers to the amount
or the dose (e.g., amount and/or interval) of drug required to
produce an intended therapeutic response. A therapeutically
effective dose refers to a dose that, as compared to a
corresponding subject who has not received such a dose, results in
improved treatment, healing, prevention, or amelioration of a
disease, disorder, or side effect, or a decrease in the rate of the
occurrence or advancement of a disease or disorder. The term also
includes within its scope, doses effective to enhance physiological
functions.
[0023] The terms "treat," "treating," and "treatment" refer to
methods of alleviating, abating, or ameliorating a disease or
symptom, preventing an additional symptom, ameliorating or
preventing an underlying cause of a symptom, inhibiting a disease
or condition, arresting the development of a disease or condition,
relieving a disease or condition, causing regression of a disease
or condition, relieving a condition caused by the disease or
condition, or stopping a symptom of the disease or condition either
prophylactically and/or after the symptom has occurred.
[0024] As used herein with reference to a particular dose,
"kg.sup.-1", "per kg", "/kg," and "per kilogram" represent "per
kilogram of body weight" of the mammal, and thus the terms can be
used interchangeably.
[0025] As used herein, the term "polypeptide" is intended to
encompass a singular "polypeptide" as well as plural
"polypeptides," and refers to a molecule composed of monomers
(amino acids) linearly linked by amide bonds (also known as peptide
bonds). The term "polypeptide" refers to any chain or chains of two
or more amino acids, and does not refer to a specific length of the
product. Thus, peptides, dipeptides, tripeptides, oligopeptides,
"proteins," "amino acid chains," or any other term used to refer to
a chain or chains of two or more amino acids, are included within
the definition of "polypeptide," and the term "polypeptide" may be
used instead of, or interchangeably with any of these terms. The
term "polypeptide" is also intended to be inclusive of the products
of post-expression modifications of the polypeptide, including
without limitation glycosylation, acetylation, phosphorylation,
amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, or modification by non-naturally occurring
amino acids. A polypeptide may be derived from a natural biological
source or produced by recombinant technology, but is not
necessarily translated from a designated nucleic acid sequence. It
may be generated in any manner, including by chemical
synthesis.
[0026] Other polypeptides disclosed herein are fragments,
derivatives, analogs, or variants of the foregoing polypeptides,
and any combination thereof. The terms "fragment," "variant,"
"derivative" and "analog" when referring to any of the polypeptides
disclosed herein include any polypeptides which retain at least
some of the activity of the corresponding native polypeptide (e.g.,
NAGLU polypeptide fragments, variants, derivatives, and analogs
that retain the ability to hydrolyze terminal non-reducing
N-acetyl-D-glucosamine residues in
N-acetyl-.alpha.-D-glucosaminides). Fragments of polypeptides
include, for example, proteolytic fragments, as well as deletion
fragments. Variants of a polypeptide include fragments as described
above, and also polypeptides with altered amino acid sequences due
to amino acid substitutions, deletions, or insertions. Variants can
occur naturally or be non-naturally occurring. Non-naturally
occurring variants can be produced using art-known mutagenesis
techniques. Variant polypeptides can comprise conservative or
non-conservative amino acid substitutions, deletions, or additions.
Derivatives are polypeptides which have been altered so as to
exhibit additional features not found on the native polypeptide.
Examples include fusion proteins. Variant polypeptides can also be
referred to herein as "polypeptide analogs." As used herein, a
"derivative" of a subject polypeptide can contain one or more
residues chemically derivatized by reaction of a functional side
group. Also included as "derivatives" are those peptides which
contain one or more naturally occurring amino acid derivatives of
the twenty standard amino acids. For example, 4-hydroxyproline can
be substituted for proline; 5-hydroxylysine can be substituted for
lysine; 3-methylhistidine can be substituted for histidine;
homoserine can be substituted for serine; and/or ornithine can be
substituted for lysine.
[0027] As used herein, the terms "glycan," "glycan structure,"
"glycan moiety," "oligosaccharide," "oligosaccharide structure,"
"glycosylation pattern," "glycosylation profile," and
"glycosylation structure" have essentially the same meaning and
each refers to one or more structures which are formed from sugar
residues and are attached to glycosylated protein such as human
NAGLU. For example, "N-glycan" or "N-linked glycan" refers to a
glycan structure attached to a nitrogen of asparagine or arginine
sidechain of the glycosylated protein. "O-glycan" or "O-linked
glycan" refers to a glycan structure attached to the hydroxyl
oxygen of serine, threonine, tyrosine, hydroxy lysine, or
hydroxyproline side chain of the glycosylate protein.
[0028] The term "polynucleotide" is intended to encompass a
singular nucleic acid as well as plural nucleic acids, and refers
to an isolated nucleic acid molecule or construct, e.g., messenger
RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may comprise a
conventional phosphodiester bond or a non-conventional bond (e.g.,
an amide bond, such as found in peptide nucleic acids (PNA)). The
term "nucleic acid" refers to any one or more nucleic acid
segments, e.g., DNA or RNA fragments, present in a polynucleotide.
An "isolated" nucleic acid or polynucleotide is one that has been
removed from its native environment. For example, a recombinant
polynucleotide encoding NAGLU contained in a vector is considered
isolated for the purposes of the present invention. Further
examples of an isolated polynucleotide include recombinant
polynucleotides maintained in heterologous host cells or purified
(partially or substantially) polynucleotides in solution. Isolated
RNA molecules include in vivo or in vitro RNA transcripts of
polynucleotides of the present invention. Isolated polynucleotides
or nucleic acids according to the present invention further include
such molecules produced synthetically. In addition, a
polynucleotide or a nucleic acid can be or can include a regulatory
element such as a promoter, ribosome binding site, or a
transcription terminator.
[0029] As used herein, a "coding region" is a portion of nucleic
acid which consists of codons translated into amino acids. Although
a "stop codon" (TAG, TGA, or TAA) is not translated into an amino
acid, it may be considered to be part of a coding region, but any
flanking sequences, for example promoters, ribosome binding sites,
transcriptional terminators, introns, and the like, are not part of
a coding region. Two or more coding regions of the present
invention can be present in a single polynucleotide construct,
e.g., on a single vector, or in separate polynucleotide constructs,
e.g., on separate (different) vectors. Furthermore, any vector can
contain a single coding region, or can comprise two or more coding
regions. In addition, a vector, polynucleotide, or nucleic acid of
the invention can encode heterologous coding regions, either fused
or unfused to a nucleic acid encoding an NAGLU polypeptide or
fragment, variant, or derivative thereof. Heterologous coding
regions include without limitation specialized elements or motifs,
such as a secretory signal peptide or a heterologous functional
domain.
[0030] A variety of transcription control regions are known to
those skilled in the art. These include, without limitation,
transcription control regions which function in vertebrate cells,
such as, but not limited to, promoter and enhancer segments from
cytomegaloviruses (the immediate early promoter, in conjunction
with intron-A), simian virus 40 (the early promoter), and
retroviruses (such as Rous sarcoma virus). Other transcription
control regions include those derived from vertebrate genes such as
actin, heat shock protein, bovine growth hormone and rabbit
.beta.-globin, as well as other sequences capable of controlling
gene expression in eukaryotic cells. Additional suitable
transcription control regions include tissue-specific promoters and
enhancers as well as lymphokine-inducible promoters (e.g.,
promoters inducible by interferons or interleukins).
[0031] Similarly, a variety of translation control elements are
known to those of ordinary skill in the art. These include, but are
not limited to ribosome binding sites, translation initiation and
termination codons, and elements derived from picornaviruses
(particularly an internal ribosome entry site, or IRES, also
referred to as a CITE sequence).
[0032] In other embodiments, a polynucleotide of the present
invention is RNA, for example, in the form of messenger RNA
(mRNA).
[0033] Polynucleotide and nucleic acid coding regions of the
present invention may be associated with additional coding regions
which encode secretory or signal peptides, which direct the
secretion of a polypeptide encoded by a polynucleotide of the
present invention. According to the signal hypothesis, proteins
secreted by mammalian cells have a signal peptide or secretory
leader sequence which is cleaved from the mature protein once
export of the growing protein chain across the rough endoplasmic
reticulum has been initiated. Those of ordinary skill in the art
are aware that polypeptides secreted by vertebrate cells generally
have a signal peptide fused to the N-terminus of the polypeptide,
which is cleaved from the complete or "full length" polypeptide to
produce a secreted or "mature" form of the polypeptide. In certain
embodiments, the native signal peptide, e.g., the
MEAVAVAAAVGVLLLAGAGGAAG (SEQ ID NO:3) signal peptide of human NAGLU
is used, or a functional derivative of that sequence that retains
the ability to direct the secretion of the polypeptide that is
operably associated with it. Alternatively, a heterologous signal
peptide (e.g., a heterologous mammalian or avian signal peptide),
or a functional derivative thereof, may be used. For example, the
wild-type leader sequence may be substituted with the leader
sequence of human tissue plasminogen activator (TPA) or mouse
.beta.-glucuronidase.
[0034] "Vector" means a polynucleotide comprised of single strand,
double strand, circular, or supercoiled DNA or RNA. A typical
vector can be comprised of the following elements operatively
linked at appropriate distances for allowing functional gene
expression: replication origin, promoter, enhancer, 5' mRNA leader
sequence, ribosomal binding site, nucleic acid cassette,
termination and polyadenylation sites, and selectable marker
sequences. One or more of these elements can be omitted in specific
applications. The nucleic acid cassette can include a restriction
site for insertion of the nucleic acid sequence to be expressed. In
a functional vector the nucleic acid cassette contains the nucleic
acid sequence to be expressed including translation initiation and
termination sites. An intron optionally can be included in the
construct, for example, 5' to the coding sequence. A vector is
constructed so that the particular coding sequence is located in
the vector with the appropriate regulatory sequences, the
positioning and orientation of the coding sequence with respect to
the control sequences being such that the coding sequence is
transcribed under the "control" of the control or regulatory
sequences. Modification of the sequences encoding the particular
protein of interest can be desirable to achieve this end. For
example, in some cases it can be necessary to modify the sequence
so that it can be attached to the control sequences with the
appropriate orientation, or to maintain the reading frame. The
control sequences and other regulatory sequences can be ligated to
the coding sequence prior to insertion into a vector.
Alternatively, the coding sequence can be cloned directly into an
expression vector which already contains the control sequences and
an appropriate restriction site which is in reading frame with and
under regulatory control of the control sequences.
[0035] The term "expression" as used herein refers to a process by
which a gene produces a biochemical, for example, a polypeptide.
The process includes any manifestation of the functional presence
of the gene within the cell including, without limitation, gene
knockdown as well as both transient expression and stable
expression. It includes without limitation transcription of the
gene into messenger RNA (mRNA), and the translation of such mRNA
into polypeptide(s). Expression of a gene produces a "gene
product." As used herein, a gene product can be either a nucleic
acid, e.g., a messenger RNA produced by transcription of a gene, or
a polypeptide which is translated from a transcript. Gene products
described herein further include nucleic acids with post
transcriptional modifications, e.g., polyadenylation, or
polypeptides with post translational modifications, e.g.,
methylation, glycosylation, the addition of lipids, association
with other protein subunits, proteolytic cleavage, and the
like.
[0036] As used herein, "host cells" refers to cells that harbor
vectors constructed using recombinant DNA techniques and encoding
at least one heterologous gene.
[0037] As used herein, the term "pharmaceutical composition" refers
to a mixture of a compound described herein with other chemical
components, such as carriers, stabilizers, diluents, dispersing
agents, suspending agents, thickening agents, and/or
excipients.
[0038] The words "preferred" and "preferably" refer to embodiments
of the invention that may afford certain benefits, under certain
circumstances. However, other embodiments may also be preferred,
under the same or other circumstances, whether described as such or
not herein. Furthermore, the recitation of one or more preferred
embodiments does not imply that other embodiments are not useful,
and is not intended to exclude other embodiments from the scope of
the invention.
[0039] The terms "comprises" and variations thereof do not have a
limiting meaning where these terms appear in the description and
claims.
[0040] Unless otherwise specified, "a," "an," "the," and "at least
one" are used interchangeably and mean one or more than one.
[0041] Also herein, the recitations of numerical ranges by
endpoints include all numbers subsumed within that range (e.g., 1
to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
[0042] For any method disclosed herein that includes discrete
steps, the steps may be conducted in any feasible order. And, as
appropriate, any combination of two or more steps may be conducted
simultaneously.
[0043] All headings are for the convenience of the reader and
should not be used to limit the meaning of the text that follows
the heading, unless so specified.
[0044] The above summary of the invention is not intended to
describe each disclosed embodiment or every implementation of the
invention. The description that follows more particularly
exemplifies illustrative embodiments. In several places throughout
the application, guidance may be provided through lists of
examples, which examples can be used in various combinations. In
each instance, the recited list serves only as a representative
group and should not be interpreted as an exclusive list.
DESCRIPTION OF THE FIGURES
[0045] FIG. 1 depicts the amino acid sequence of wild-type human
N-acetyl-.alpha.-D-glucosaminidase (NAGLU) (SEQ ID NO:1). Amino
acid residues 1-23 represent a signal peptide. See UniProtKB P54802
ANAG_Human.
[0046] FIG. 2 depicts cDNA of wild-type human NAGLU (SEQ ID
NO:2).
[0047] FIG. 3 depicts the location of mutations for NAGLU variants
on a NAGLU protein. Mutation positions for the eight novel variants
are marked by red dots. Bars show the three conserved domains in
NAGLU: N-terminal domain, TIM barrel domain, and C-terminal
domain.
ILLUSTRATIVE DESCRIPTION OF THE INVENTION
[0048] This disclosure describes mutations in the nucleotide
sequence of a human NAGLU gene that may be associated with reduced
N-acetyl-.alpha.-D-glucosaminidase (NAGLU) activity. NAGLU
activity-reducing mutations are of clinical and research interest
because reduced NAGLU activity is a marker for MPS IIIB. It is
expected that the identification of NAGLU activity-reducing
mutations in the human NAGLU gene as set forth herein will be of
important medical significance in that it will improve genetic
screening for human subjects suspected of having MPS TIM.
[0049] In one aspect, the invention provides a method for detecting
a potentially pathogenic mutation in a human NAGLU nucleotide
sequence. The detection method includes providing a biological
sample that includes a nucleic acid; and performing a genetic
analysis on the biological sample to detect the presence of a
potentially pathogenic mutation in a human NAGLU nucleotide
sequence. Optionally, the detection method includes diagnosing a
patient with MPS IIIB when the presence of a potentially pathogenic
mutation in the NAGLU nucleotide sequence of the patient is
detected. The method may further include administering a
therapeutically effective amount of a recombinant human NAGLU to
the patient. An exemplary recombinant human NAGLU that can be
administered to a patient is described in WO2013/055888 and US
2013/0095092.
[0050] In another aspect, the invention provides a method for
diagnosing MPS IIIB in a patient. The diagnostic method includes
providing a biological sample from a patient, wherein the sample
includes a nucleic acid; performing a genetic analysis on the
biological sample to detect the presence of a potentially
pathogenic mutation in a human NAGLU nucleotide sequence; and
diagnosing the patient with MPS IIIB when the presence of a
potentially pathogenic mutation in the NAGLU nucleotide sequence of
the subject is detected. The method may further include
administering a therapeutically effective amount of a recombinant
human NAGLU to the patient.
[0051] In another aspect, the invention provides a method for
treating a patient afflicted with or suspected of being afflicted
with MPS IIIB. The method includes administering a therapeutically
effective amount of a recombinant human NAGLU to the patient who
has been determined to possess at least one NAGLU allelic variant
that includes a potentially pathogenic mutation as described
herein. Both NAGLU alleles of the patient may have pathogenic
mutations; in other words, the patient may have biallelic
pathogenic variants in the NAGLU gene.
[0052] The potentially pathogenic mutation can include, for
example, a nucleotide sequence mutation encoding an amino acid
substitution at amino acid position L35, M338, R541, and/or L622
relative to the wild-type human NAGLU amino acid sequence (SEQ ID
NO:1), and/or, for example, a nucleotide substitution or in frame
deletion such as c.104T>C, c.1012A>G, c.1621C>T,
c.1865T>C, or c.1700_1708del9 relative to the wild-type human
NAGLU cDNA sequence (SEQ ID NO:2), where "c." designates numbering
according to the coding sequence. Alternatively or additionally,
the potentially pathogenic mutation can include one or more
mutations that affect precursor mRNA splicing, and/or that cause a
frame shift or truncation, such as c.1128_1138del11, c.351delG, and
IVS2+5G>A relative to the wild-type human NAGLU cDNA sequence
(SEQ ID NO:2). In some embodiments, the mutation is a NAGLU
activity-reducing mutation.
[0053] In one embodiment, a potentially pathogenic mutation in the
NAGLU gene is identified by analyzing a biological sample obtained
from a patient known to be afflicted with, or suspected of being
afflicted with, MPS IIIB. NAGLU gene variants that include one or
more potentially pathogenic mutations can be clinically identified
by performing genetic analysis on a biological sample obtained from
a such a patient, where the biological sample to be analyzed
contains a nucleic acid, for example genomic DNA or RNA.
Illustrative examples of novel, clinically identified potentially
pathogenic mutations in the NAGLU gene are set forth in Example I.
Clinically identified mutations are of particular clinical and
research interest because they are discovered in patients who have
been diagnosed with (or are suspected of having) MPS IIIB. It is
expected that the identification of these mutations in the
nucleotide sequence of a human NAGLU gene will be of important
medical significance in that it will improve genetic screening for
human subjects suspected of having MPS IIIB.
[0054] Genetic analysis of a biological sample can be performed,
for example, using whole transcriptome sequencing, whole exome
sequencing, whole genome sequencing, or hybridization to a DNA
microarray, as described further elsewhere herein. In methods that
involve the use of a biological sample, the biological sample can
be obtained from a pediatric or adult patient.
[0055] It should be understood that mutations in the NAGLU gene of
a patient, including pathogenic mutations and potentially
pathogenic mutations, can be present in one or both NAGLU alleles
of the patient. Pathogenic mutations may be present in both alleles
of a patient diagnosed with MPS TIM, as MPS TIM is an autosomal
recessive disorder. In other words, the patient may have biallelic
pathogenic variants of the NAGLU gene A NAGLU allele of a patient
may contain one or more pathogenic or potentially pathogenic
mutations, which mutations may be the same as or different from the
mutations, if any, present in the other NAGLU allele.
[0056] In another embodiment, a potentially pathogenic mutation in
the NAGLU gene is identified by analyzing NAGLU nucleotide or amino
acid sequence data available from various protein or nucleic acid
databases. As shown in Table 2, over 200 NAGLU gene variants are
known. The coding region of a NAGLU gene variant described in a
database can be genetically engineered into a cellular or acellular
expression system, and the expressed protein can be assayed for
enzymatic activity, for example using an in vitro enzyme assay, as
described in more detail elsewhere herein and as exemplified in
Example II. A cellular assay can be used, although an in vitro
assay is preferred. The method permits evaluation of any known
human NAGLU nucleotide sequence, such as those variants cataloged
in the Exome Aggregation Consortium (ExAC) database, for potential
pathogenicity. As explained in more detail elsewhere herein, NAGLU
gene variants identified in this manner may be considered
potentially pathogenic if the enzyme encoded by the NAGLU gene
variant exhibits reduced NAGLU activity.
[0057] The invention further provides a method of screening for
presence of MPS IIIB in a patient. The method includes determining
the nucleotide sequence of one or more of the patient's NAGLU
alleles; and determining whether one or more of the patient's NAGLU
alleles includes a potentially pathogenic mutation as described
herein. Optionally, the screening method includes screening for the
presence of additional disease in the patient; for example, the
screening method can include screening for one or more additional
Mendelian disorders.
[0058] In another aspect, the invention provides a kit for
detecting a potentially pathogenic mutation in a human NAGLU
nucleotide sequence. In one embodiment, the kit includes at least
one oligonucleotide primer specific for a potentially pathogenic
mutation as described herein, and optionally, instructions relating
to detecting mutations in the NAGLU nucleotide sequence. In another
embodiment, the kit includes at least one allele-specific
oligonucleotide probe for a potentially pathogenic mutation as
described herein, and instructions relating to detecting mutations
in the NAGLU nucleotide sequence.
[0059] In another aspect, the invention provides an isolated
polynucleotide that includes a mutated human NAGLU nucleotide
sequence having a mutation selected from the group consisting of
c.104T>C, c.1012A>G, c.1621C>T, c.1865T>C,
c.1128_1138del11, c.351delG, IVS2+5G>A, and c.1700_1708del9
relative to the wild-type NAGLU nucleotide coding sequence (SEQ ID
NO:2), as well as a vector operably encoding the polynucleotide,
and a host cell that includes the vector. Methods of making and
using the polynucleotide, vector and host cell are also encompassed
by the invention.
Reduction in NAGLU Activity
[0060] NAGLU is enzyme that has N-acetyl-.alpha.-D-glucosaminidase
activity. "Reduced NAGLU activity" is defined in relation to the
N-acetyl-.alpha.-D-glucosaminidase activity of wild-type human
NAGLU. The activity of the gene products by NAGLU variants can be
measured in an assay that supplies 4-methylumbelliferyl
N-acetyl-.alpha.-D-glucosaminide (4MU-NAG) as a substrate, and
detects the production of the cleaved fluorophore
4-methylumbelliferyl at excitation/emission wavelengths of
360/460+/-40 nm. A representative activity assay is described in
Example II. In the cell lysate assay described in Example II, a
NAGLU gene product can be considered as having "reduced NAGLU
activity" if, for example, it exhibits less activity than of
wild-type NAGLU activity. In an assay of cell culture supernatant
described in Example II, a NALGU gene product is considered as
having "reduced NAGLU activity" if, for example, it exhibits
significantly less activity than of wild-type NAGLU activity.
Cutoff values can be used to determine reduced NAGLU activity.
Exemplary cutoff values for cell lysate activity of 1%, 2%, 5%, 7%
10%, 12% 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85% or higher, or any value in between, can be used
to identify allelic variants having "reduced NAGLU activity."
Likewise, an exemplary cutoff value for supernatant activity of
0.1%, 0.2%, 0.5%, 0.7% 1.0%, 1.2% 1.5%, 2.0%, 2.5%, 3.0%, 3.5%,
4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 10%,
12%, 15%, 20%, 25%, 30%, 35%, 40% or higher, or any value in
between, can be used to identify allelic variants having "reduced
NAGLU activity." The invention is not limited by the activity assay
used, and other activity assays can be employed. See, e.g., Mauri
et al., PLoS One, (2013) 8(6):e60860; Marsh et al., Clinical
Genetics (1985) 27: 258-262; Chow et al., Carbohydrate Research
(1981) 96:87-93; Weber et al., Protein Expression and Purification,
(2001)21:251-259); WO2013/055888. Allelic variants that exhibited
reduced NAGLU activity are characterized herein as possessing
"NAGLU activity-reducing" mutations. The characteristic of "reduced
NAGLU activity" is used to identify mutant NAGLU gene products that
may be considered potentially pathogenic NAGLU variants and thereby
indicative of or associated with MPS TIM.
[0061] It should be understood that NAGLU gene products exhibiting
"reduced NAGLU activity" include those gene products having no
detectable NAGLU activity. Gene products having no detectable NAGLU
activity may have activity at too low a level to be detected, or
they may have no activity at all (i.e., activity has been
eliminated).
NAGLU Activity-Reducing Mutations
[0062] FIG. 1 shows the amino acid sequence of wild-type (normal)
human NAGLU (SEQ ID NO:1). FIG. 2 shows a sequence of the coding
region of the wild-type (normal) human NAGLU gene (SEQ ID NO:2).
The newly identified NAGLU activity-reducing mutations are
described with reference to the human NAGLU wild-type (normal)
sequence. It should be understood that one or more of the NAGLU
activity-reducing mutations described herein may occur alone,
together, and/or in combination with one or more known mutations in
the human NAGLU gene.
[0063] A mutation in the nucleotide sequence of the human NAGLU
gene can, in some embodiments, result in an amino acid substitution
in the NAGLU encoded by the mutated NAGLU gene. In some
embodiments, the mutation causes a truncation of NAGLU. The
mutation can be, for example, a missense mutation or a nonsense
mutation. In some embodiments, the mutation causes misregulation of
precursor RNA splicing, and/or alternative precursor mRNA splicing.
In some embodiments, the mutation can include an insertion or
deletion and may or may not cause a frameshift in the NAGLU encoded
by the mutated NAGLU gene. A mutation can occur in an exon or an
intron; a mutation in an intron may not alter the NAGLU coding
sequence, but may, for example, affect splicing. Potentially
pathogenic gene variants may therefore include single base, site
specific, or other types of nucleotide sequence mutations.
[0064] In some embodiments, a mutation in the nucleotide sequence
of the human NAGLU gene can occur in a region that encodes a NAGLU
protein domain, such as an N-terminal domain, a TIM barrel domain,
and/or a C-terminal domain. Potentially pathogenic NAGLU gene
variants can include variants having mutations in or affecting one
or more NAGLU protein domain, including but not limited to an
N-terminal domain, a TIM barrel domain, a C-terminal domain, or any
combination thereof, including one or more mutations that span or
affect more than one protein domain.
[0065] In some embodiments, NAGLU activity-reducing mutations in a
NAGLU gene include amino acid substitutions or in frame deletions
at one or more of the amino acid positions in the encoded NAGLU
gene product as shown in Table 1 and/or in Table 2. Exemplary
mutations producing potentially pathogenic gene variants can
include amino acid substitutions at one or more of positions L35,
M338, R541, or L622, or in frame deletion at A567_Q569del, as
numbered for human wild-type NAGLU in FIG. 1, and/or any of the
positions shown in Table 1 and/or Table 2.
[0066] When screening a subject's DNA or RNA for possible
pathogenic NAGLU allelic variants according to the method of the
invention, one of skill in the art can readily determine by
inspecting SEQ ID NO:2 (the wild-type human cDNA sequence for
NAGLU) which nucleotide base changes relative to the wild-type cDNA
sequence will yield amino acid substitutions. Nucleotide changes
that will generate an amino acid substitution at a particular
position can be determined with reference to the codon encoding the
amino acid at that position in the normal NAGLU sequence, and there
are only a limited number of possibilities permitted by the genetic
code. Exemplary amino acid substitutions or in frame deletions
yielding a potentially pathogenic mutant NAGLU are shown in Table 1
and/or Table 2 and/or include the following: L35P, M338V, R541W,
L622P, and A567_Q569del.
[0067] In some embodiments, NAGLU activity-reducing mutations in a
NAGLU gene include mutations that affect precursor mRNA splicing,
and/or that include frame shift mutations or truncations. Exemplary
mutations producing potentially pathogenic gene variants can
include frameshifts or splicing variants such as such
c.1128_1138del11, c.351delG, and IVS2+5G>A, and/or other
mutations shown in Table 1 and/or Table 2.
TABLE-US-00001 TABLE 1 Clinically identified pathogenic mutations
Amino Exemplary Amino Mutation Acid Position* Acid Mutation c.104T
> C L35 L35P c.1012A > G M338 M338V c.1621C > T R541 R541W
c.1865T > C L622 L622P c.1128_1138del11 (frameshift) c.351delG
(frameshift) IVS2 + 5G > A (splice variant) c.1700_1708del9 (in
frame deletion) A567_Q569del *Position as specified in wild-type
human NAGLU (SEQ ID NO: 1)
[0068] The newly identified NAGLU activity-reducing mutations
identified herein can occur alone or in combination with each other
or with one or more known pathogenic mutations in the nucleotide
sequence of the human NAGLU gene. Exemplary known amino acid
substitutions resulting in pathogenic allelic variants are included
in Table 2. Exemplary known frame shift mutations resulting in
pathogenic allelic variants are also included in Table 2. Table 2
also includes mutation information for allelic NAGLU variants that
have not yet been analyzed for pathogenicity, but can be analyzed
in accordance with the method described herein for assaying NAGLU
activity in vitro and determining potential pathogenicity. Table 2
also includes some of the newly identified pathogenic mutations
identified in Example I.
In Silico Analysis
[0069] In some embodiments, the potential pathogenicity of a NAGLU
gene variant can be evaluated using in silico techniques. One or
more in silico prediction methods, such as MutationTaster,
PolyPhen2, SIFT, and/or Provean, are utilized to assess the
severity of missense mutations and to predict the impact of the
mutation on NAGLU enzymatic activity. Missense mutations can be
identified based on a unanimous score, or on a consensus score, of
two, three, four or more in silico methods. For example, a mutation
can be identified as "deleterious" if four methods predict it to be
"deleterious."
NAGLU Gene Variant Detection
[0070] The invention includes a method for detecting the presence
or absence of an NAGLU activity-reducing mutation or a novel
clinically identified mutation in the nucleotide sequence of a
NAGLU gene of a human subject. Any one or more of the NAGLU
activity-reducing mutations or the novel clinically identified
mutations described herein can be detected. The presence of one or
more NAGLU alleles possessing an NAGLU activity-reducing mutation
or clinically identified mutation as described herein (i.e., one or
more potentially pathogenic NAGLU gene variants) has clinical
relevance to the diagnosis of MPS IIIB.
[0071] Detection of a mutation in the nucleotide sequence of a
NAGLU gene can be accomplished using any convenient method. Many
techniques for genetic testing and genetic analysis are known to
the art. Genetic analysis for the presence of a potentially
pathogenic NAGLU gene variant in a subject can be carried out, for
example, using recently developed nucleotide sequencing
technologies or using traditional hybridization technologies. For
example, the analysis can be carried out using positional cloning
based on linkage analysis and/or Sanger sequencing. Another option
is RNA whole transcriptome sequencing, which may also be referred
to as RNA sequencing, RNA-seq or whole transcriptome shotgun
sequencing (WTSS), which typically utilizes next-generation
sequencing technologies (NGS) and focuses on a gene expression
profile, is able to detect alternative splicing events, and can
detect single nucleotide variants. Another option is exome
sequencing or whole exome sequencing (WES or WXS), wherein some or
all of the expressed genes in a genome (i.e., the exome) are
sequenced. Whole-exome sequencing facilitates identification of
autosomal recessive disease genes in single patients from
non-consanguineous families. Another option is whole-genome
sequencing (WGS) which provides a complete view of the human
genome, including point mutations in distant enhancers and other
regulatory elements. Pabinger et al., Brief. Bioinform (2014)
15(2):256-278, Epub Jan. 21, 2013. Other exemplary methods of
genetic analysis include, but are not limited to, restriction
fragment length polymorphism identification (RFLPI) of genomic DNA,
random amplified polymorphic detection (RAPD) of genomic DNA,
amplified fragment length polymorphism detection (AFLPD),
polymerase chain reaction (PCR), DNA sequencing, allele specific
oligonucleotide (ASO) probes, and hybridization to DNA microarrays
or beads. It should be understood that mutation analysis and
detection techniques are rapidly evolving and detection of a
potentially pathogenic NAGLU gene variant is not limited to any
particular detection technique.
[0072] Mutations in the nucleotide sequence of a NAGLU gene can be
detected by analyzing any polynucleotide or polynucleotide fragment
that derives from, directly or indirectly the NAGLU gene of a
subject. Types of polynucleotides that can be analyzed for
mutations include, without limitation, genomic DNA (whole or
partial), exomic RNA (whole or partial), primary RNA transcripts
such as precursor RNA, processed RNA such as spliced mRNA, mature
RNA and mRNA, and cDNA. The nucleotide sequence to be analyzed
typically includes coding sequences, for example exon sequences,
and may also include intron sequences, particularly intron
sequences that proximal to splice junctions and may thus affect
production of the mature mRNA and/or protein translation and
structure.
[0073] The nucleic acids (e.g., DNA or RNA) to be analyzed for
presence of one or more NAGLU activity-reducing and/or novel
clinically identified gene mutations can be present in or isolated
from a biological sample obtained from the subject. The biological
sample can be a tissue sample or a fluid sample, for example. The
subject can be a child or an adult. Nucleic acids may be fragmented
into smaller constituent polynucleotides, prior to analysis.
[0074] Advantageously, if the detection method yields a positive
result, in that one or more of the specified pathogenic NAGLU
allelic variants is detected in a subject, genetic testing can then
be performed on blood relatives in order to determine whether other
family members possess the potentially pathogenic NAGLU allelic
variants.
Diagnostic Methods
[0075] Detection of a potentially pathogenic NAGLU gene variant in
a human subject can aid in making, or can confirm, a diagnosis of
MPS MB in the subject. Lysosomal storage diseases that can be
diagnosed using the diagnostic method of the invention include MPS
IIIB. The nucleotide sequence of NAGLU gene of the subject, or
associated mRNA or cDNA, is analyzed as in the detection method in
order to determine the presence of a mutation associated with
reduced NAGLU activity, wherein reduced NAGLU activity of the gene
variant is consistent with or indicative of MPS IIIB, and or a
clinically identified mutation in the nucleotide sequence of a
NAGLU gene as described herein. Optionally, the diagnostic method
includes determining whether other signs or symptoms associated
with MPS IIIB are present in the subject. This determination can be
made before or after genetic analysis to determine whether the
subject carries a potentially pathogenic NAGLU gene variant. The
diagnostic method of the invention, involving the detection of
genetic mutations, can also optionally be performed in combination
with or as an adjunct to, either before or after, one or more
assays for deficient NAGLU enzyme activity in peripheral blood
leukocytes, fibroblasts, or dried blood spots. Thus, the diagnostic
method of the invention optionally further includes measuring NAGLU
activity in the subject. The level of NAGLU activity in a patient
prior to treatment can be about 1%, about 2%, about 3%, about 5%,
about 10%, about 15%, about 20%, about 30%, about 40%, about 50%,
about 60%, about 70%, or about 80% of normal levels of NAGLU
activity. For example, the level of NAGLU activity in a patient
prior to treatment can be about 50% or less of normal levels of
NAGLU activity, or about 40% or less of normal levels of NAGLU
activity, or about 30% or less of normal levels of NAGLU activity,
or about 20% or less of normal levels of NAGLU activity, or about
10% or less of normal levels of NAGLU activity, or about 5% or less
of normal levels of NAGLU activity. Some patients show no
measurable NAGLU activity prior to treatment. The level of NAGLU
activity in a patient can be measured in cultured fibroblasts or
lymphocytes (e.g., leukocytes) obtained from a human patient
suffering from NAGLU deficiency. Lymphocytes include, but are not
limited to, peripheral blood mononuclear cells (PMBC). Enzymatic
activity can be measured using methods known in the art, for
example according to the methods described in Mauri et al., PLoS
One, (2013) 8(6):e60860; Marsh et al., Clinical Genetics (1985) 27:
258-262, Chow et al., Carbohydrate Research (1981) 96:87-93; and
Weber et al., Protein Expression and Purification,
(2001)21:251-259).
[0076] The diagnostic method can be performed on a subject
suspected of having MPS IIIB, or as a component of a more general
genetic screen. For example, a subject can be screened for the
presence of a potentially pathogenic NAGLU allelic variant in
combination with screening for pathogenic gene variants associated
with other Mendelian disorders such as phenylketonuria, cystic
fibrosis, sickle-cell anemia, oculocutaneous albinism, Huntington's
disease, myotonic dystrophy, hypercholesterolemia,
neurofibromatosis, polycystic kidney disease, hemophilia,
Duchenne's muscular dystrophy, Rett's syndrome, or other Mendelian
diseases (see the National Center for Biotechnology Information
(NCBI) databases Online Mendelian Inheritance in Man,
http://www.ncbi.nlm.nih.gov/omim, and ClinVar,
http://www.ncbi.nlm.nih.gov/omim; see also Chial et al., Nature
Education 1(1):192 (2008)).
[0077] The diagnostic method of the invention optionally includes
treating the patient for MPS IIIB.
Treatment Methods
[0078] The invention also includes treating a patient who has been
found to carry a potentially pathogenic NAGLU variant as described
herein. Such a patient is either afflicted with, or suspected of
being afflicted with MPS IIIB. In some embodiments, the patient is
treated with enzyme replacement therapy using an exogenous NAGLU,
for example by using recombinant human NAGLU (rhNAGLU). A patient
who has been found to carry a potentially pathogenic NAGLU variant
can be treated by administering a therapeutically effective amount
of exogenous NAGLU. The exogenous NAGLU can be a recombinant human
NAGLU such as described in WO2013/055888 and US 2013/0095092, and
may have an N-linked glycan structure that includes at least one
mannose and/or mannose-6-phosphate.
[0079] The method for treating a patient who has been found to
carry a potentially pathogenic NAGLU variant can include can
include single or multiple administrations of a therapeutically
effective amount of exogenous NAGLU. Exogenous NAGLU can be
administered at regular intervals, depending on the nature,
severity and extent of the subject's condition. In some
embodiments, a therapeutically effective amount of exogenous NAGLU
may be administered intravenously or intrathecally, periodically at
regular intervals, e.g., once every year, once every six months,
once every five months, once every three months, bimonthly (once
every two months), monthly (once every month), biweekly (once every
two weeks), weekly, or more frequently, such as every day or every
other day. The exogenous NAGLU may be effectively internalized into
human cells, and may cause an increase in NAGLU activity in the
patient. An increase in NAGLU activity can be measured, for
example, in vitro or in human cells such as lymphocytes and/or
fibroblasts as described herein and elsewhere, such as in
WO2013/055888 and US 2013/0095092.
[0080] In one embodiment, exogenous NAGLU is administered
intravenously to the subject at a dosage of about 0.5 to about 50
mg/kg body weight. In another embodiment, exogenous NAGLU is
administered intravenously to the subject at a dosage of about 1 to
about 30 mg/kg body weight. In another embodiment, exogenous NAGLU
is administered intravenously to the subject at a dosage of about 6
to about 27 mg/kg body weight.
[0081] In one embodiment, an effective amount of exogenous NAGLU is
administered intravenously to a subject in need thereof every other
week (QOW). In some aspects, the method includes administering a
dose of about 0.3 mg/kg to about 10 mg/kg of exogenous NAGLU every
other week. For example, a dose of about 0.3 mg/kg, about 1 mg/kg,
about 3 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8
mg/kg, about 9 mg/kg, or about 10 mg/kg, can be administered every
other week. Preferably, the intravenous administration is by
intravenous infusion, such as a two hour intravenous infusion.
[0082] In some embodiments, exogenous NAGLU is administered by
infusion, and the infusion can occur over an extended time period,
for example, 30 minutes to 10 hours. Thus, the infusion can occur,
for example, over a period of about 1 hour, about 2 hours, about 3
hours, about 4 hours, or about 5 hours. The infusion can also occur
at various rates. Thus, for example, the infusion rate can be about
1 mL per hour to about 20 mL per hour. In some embodiments, the
infusion rate is 5 mL to 10 mL per hour. In one embodiment, the
infusion rate is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19 or 20 mL per hour. In one embodiment, the infusion
rate is 0.1 to 5 mg/kg/hr. In one embodiment, the infusion rate is
about 0.1, about 0.2, about 0.3, about 0.5, about 1.0, about 1.5,
about 2.0, about 3.0, or about 4.0 mg/kg/hr. Ranges and values
intermediate to the above recited ranges and values are also
contemplated to be part of the invention.
[0083] In one embodiment, exogenous NAGLU is administered
intravenously by IV infusion by any useful method. In one example,
exogenous NAGLU can be administered by intravenous infusion through
a peripheral line. In another example, exogenous NAGLU can be
administered by intravenous infusion through a peripherally
inserted central catheter. In another example, exogenous NAGLU can
be administered by intravenous infusion facilitated by an
ambulatory infusion machine attached to a venous vascular access
port. In one embodiment of intravenous infusion, the medication is
administered over a period of 1 to 8 hours depending on the amount
of medication to be infused and the patient's previous
infusion-related reaction history, as determined by a physician
skilled in the art. In another embodiment, exogenous NAGLU is
administered intravenously by IV injection.
[0084] In another embodiment, exogenous NAGLU can be administered
via intraperitoneal or intrathecal injection.
[0085] In another embodiment, exogenous NAGLU is intrathecally
administered to the subject. In one embodiment, the exogenous NAGLU
is intrathecally administered at a dosage of at least about 0.3,
0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 mg/kg body weight. In another
embodiment, exogenous NAGLU is intrathecally administered at a
dosage of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg body weight.
In another embodiment, exogenous NAGLU is administered
intrathecally at a dosage of about 10 to about 30 mg/kg body
weight.
[0086] In some embodiments, the therapeutically effective dose
ranges from about 0.005 mg/kg body weight to 500 mg/kg body weight,
e.g., from about 0.005 mg/kg body weight to 400 mg/kg body weight,
from about 0.005 mg/kg body weight to 300 mg/kg body weight, from
about 0.005 mg/kg body weight to 200 mg/kg body weight, from about
0.005 mg/kg body weight to 100 mg/kg body weight, from about 0.005
mg/kg body weight to 90 mg/kg body weight, from about 0.005 mg/kg
body weight to 80 mg/kg body weight, from about 0.005 mg/kg body
weight to 70 mg/kg body weight, from about 0.005 mg/kg body weight
to 60 mg/kg body weight, from about 0.005 mg/kg body weight to 50
mg/kg body weight, from about 0.005 mg/kg body weight to 40 mg/kg
body weight, from about 0.005 mg/kg body weight to 30 mg/kg body
weight, from about 0.005 mg/kg body weight to 25 mg/kg body weight,
from about 0.005 mg/kg body weight to 20 mg/kg body weight, from
about 0.005 mg/kg body weight to 15 mg/kg body weight, from about
0.005 mg/kg body weight to 10 mg/kg bra body in weight. Ranges and
values intermediate to the above recited ranges and values (e.g.,
10-50 mg/kg, 1-5 mg/kg, 2-8 mg/kg, 5-10 mg/kg, 0.1-10 mg/kg, 0.3-30
mg/kg, 0.3-50 mg/kg, 0.5-10 mg/kg, 5-30 mg/kg, or 6-27 mg/kg) are
also contemplated to be part of the invention.
[0087] In some embodiments, the therapeutically effective dose is
greater than or at least about 0.1 mg/kg body weight, greater than
or at least about 0.2 mg/kg body weight, greater than or at least
about 0.3 mg/kg body weight, greater than or at least about 0.4
mg/kg body weight, greater than or at least about 0.5 mg/kg body
weight, greater than or at least about 1.0 mg/kg body weight,
greater than or at least about 3 mg/kg body weight, greater than or
at least about 5 mg/kg body weight, greater than or at least about
6 mg/kg body weight, greater than or at least about 7 mg/kg body
weight greater than or at least about 10 mg/kg body weight, greater
than or at least about 15 mg/kg body weight, greater than or at
least about 20 mg/kg body weight, greater than or at least about 30
mg/kg body weight, greater than or at least about 40 mg/kg body
weight, greater than or at least about 50 mg/kg body weight,
greater than or at least about 60 mg/kg body weight, greater than
or at least about 70 mg/kg body weight, greater than about or at
least 80 mg/kg body weight, greater than or at least about 90 mg/kg
body weight, greater than or at least about 100 mg/kg body weight.
Ranges and values intermediate to the above recited ranges and
values are also contemplated to be part of the invention.
[0088] In some embodiments, the therapeutically effective dose may
also be defined by mg/kg brain weight. As one skilled in the art
would appreciate, the brain weights and body weights can be
correlated (see, e.g., Dekaban AS. "Changes in brain weights during
the span of human life: relation of brain weights to body heights
and body weights," Ann Neurol 1978; 4:345-56).
[0089] In some embodiments, the therapeutically effective dose may
also be defined by mg/15 cc of CSF. As one skilled in the art would
appreciate, therapeutically effective doses based on brain weights
and body weights can be converted to mg/15 cc of CSF. For example,
the volume of CSF in adult humans is approximately 150 mL (Johanson
C E, et al. "Multiplicity of cerebrospinal fluid functions: New
challenges in health and disease," Cerebrospinal Fluid Res. 2008
May 14; 5: 10). Therefore, single dose injections of 0.1 mg to 50
mg protein to adults would be approximately 0.01 mg/15 cc of CSF
(0.1 mg) to 5.0 mg/15 cc of CSF (50 mg) doses in adults.
[0090] It is to be further understood that for any particular
subject, specific dosage regimens should be adjusted over time
according to the individual need and the professional judgment of
the person administering or supervising the administration of the
enzyme replacement therapy and that dosage ranges set forth herein
are exemplary only and are not intended to limit the scope or
practice of the claimed invention.
[0091] Exemplary administration and dosing protocols, as well as
suitable pharmaceutical compositions, are exemplified in
WO2013/055888, U.S. Pat. Pub. US2013/0095092, and WO/2016/054025.
For example, WO/2016/054025 describes methods for the initial
dosing regimen and subsequent maintenance of therapeutic levels of
exogenous NAGLU in the central nervous system (e.g., brain tissue)
in a mammal undergoing long-term enzyme replacement therapy with
diseases associated with NAGLU deficiency, e.g., MPS IIIB, using
intravenous administration of exogenous NAGLU. Enzyme replacement
dosing regimens identified for treating lysosomal acid lipase
deficiency (see, e.g., U.S. Pat. No. 8,663,631, WO2012/050695, U.S.
Pat. Pub. US20130209436, and WO2011/133960) may also be applicable
to enzyme replacement therapy utilizing exogenous NAGLU.
[0092] Other treatment methods include the administration of
therapeutic polynucleotides analogous to the methods described in
US2014/0155475 for treatment of lysosomal acid lipase
deficiency.
[0093] Optionally, the patient is treated with a second
therapeutic. The second therapeutic can include, for example, a
cholesterol-reducing drug (e.g., statin or ezetimibe), an
antihistamine (e.g., diphenhydramine), or an immunosuppressant.
Nonlimiting examples of antihistamines include antihistamines
include, without limitation, clemastine, doxylamine, loratidine,
desloratidine, fexofenadine, pheniramine, cetirizine, ebastine,
promethazine, chlorpheniramine, levocetirizine, olopatadine,
quetiapine, meclizine, dimenhydrinate, embramine, dimethidene, and
dexchloropheniramine. Nonlimiting examples of immunosuppressants
include antihistamines, corticosteroids, sirolimus, voclosporin,
ciclosporin, methotrexate, IL-2 receptor directed antibodies,
T-cell receptor directed antibodies, TNF-alpha directed antibodies
or fusion proteins (e.g., infliximab, etanercept, or adalimumab),
CTLA-4-Ig (e.g., abatacept) and anti-OX-40 antibodies.
[0094] Nonlimiting examples of cholesterol-reducing drugs include
examples of such agents include: atorvastatin (Lipitor.RTM. and
Torvast.RTM., fluvastatin (Lescol.RTM.), lovastatin (Mevacor.RTM.,
Altocor.RTM., Altoprev.RTM.), pitavastatin (Livalo.RTM.,
Pitava.RTM.), pravastatin (Pravachol.RTM., Selektine.RTM.,
Lipostat.RTM.), rosuvastatin (Crestor.RTM.), and simvastatin
(Zocor.RTM., Lipex.RTM.). Suitable second therapeutics are also
described in WO2013/055888, US2013/0095092, and WO/2016/054025.
Expression of Variant NAGLU Nucleotide Sequences
[0095] As will be appreciated by a person of skill in the art, a
nucleotide sequence encoding an NAGLU can be introduced into and
optionally expressed in a host cell, and the invention encompasses
methods for introducing NAGLU-encoding nucleotide sequences into
host cells and optionally expressing them. Introducing a
potentially pathogenic NAGLU gene variant nucleotide sequence into
a host cell and optionally expressing a NAGLU encoded by the
variant can be achieved through any of a number of molecular
biology techniques. Typically, the polynucleotide encoding the
NAGLU is introduced into the cell using a vector. The vector can be
cloning vector, a shuttle vector, or an expression vector,
depending on the intended purpose. The polynucleotide may be
circular or linear, single-stranded or double stranded, and can be
DNA, RNA, or any modification or combination thereof. The vector
can be any molecule that may be used as a vehicle to transfer
genetic material into a host cell. Examples of vectors include
plasmids, viral vectors, cosmids, and artificial chromosomes,
without limitation. Examples of molecular biology techniques used
to transfer nucleotide sequences into a microorganism include,
without limitation, transfection, electroporation, infection,
transduction, and transformation. These methods are routine and
known in the art. Insertion of a vector into a target cell is
usually called transformation for bacterial cells and transfection
for eukaryotic cells, however insertion of a viral vector is often
called transduction. The terms transformation, transfection,
infection, and transduction, for the purpose of the present
invention, are used interchangeably herein.
[0096] An "expression vector" or "expression construct" is any
vector that is used to introduce a specific polynucleotide into a
target cell such that once the expression vector is inside the
cell, the protein that is encoded by the polynucleotide is produced
by the cellular transcription and translation machinery. The
expressed protein is referred to herein as "operably encoded" by
the expression vector. Typically, an expression vector includes
regulatory sequences operably linked to the polynucleotide encoding
the desired enzyme. Regulatory sequences are common to the person
of the skill in the art and may include for example, an origin of
replication, a promoter sequence, and/or an enhancer sequence. An
expression vector may include a ribosome binding site (RBS) and a
start site (e.g., the codon ATG) to initiate translation of the
transcribed message to produce the polypeptide. A vector may also
include a termination sequence to end translation.
[0097] The polynucleotide encoding the desired enzyme can exist
extrachromosomally or can be integrated into the host cell
chromosomal DNA. Typically, extrachromosomal DNA is maintained
within the vector on which it was introduced into the host cell. In
many instances, it may be beneficial to select a high copy number
vector in order to maximize the expression of the enzyme. The host
cell can be a prokaryotic or eukaryotic host cell. An exemplary
host cell is Expi293F as described in Example II. Alternative
methods of cloning, amplification, expression, and purification
will be apparent to the skilled artisan. Representative methods are
disclosed in Sambrook, Fritsch, and Maniatis, Molecular Cloning, a
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory
(1989).
[0098] The method of the invention further includes expressing the
gene product from an uncharacterized NAGLU gene variant in a host
cell, and evaluating NAGLU expression levels in order to determine
whether the NAGLU gene variant is potentially pathogenic, as
exemplified in the Examples.
Kits
[0099] Also provided is a kit for detecting an NAGLU
activity-reducing mutation or a novel clinically identified
mutation in the nucleotide sequence of a human NAGLU gene. In one
embodiment, the kit includes at least one oligonucleotide primer
specific for an NAGLU activity-reducing NAGLU gene mutation or
clinically identified mutation as described herein. In another
embodiment, the kit includes at least one allele-specific
oligonucleotide probe for an NAGLU activity-reducing NAGLU gene
mutation or a clinically identified mutation as described herein.
Optionally, the kit includes instructions relating to detecting
mutations in the NAGLU gene.
[0100] Advantageously, the kit can contain primers or probes of
sufficient number and variety so as to screen for a multiplicity of
mutations in the nucleotide sequence of the human NAGLU gene,
thereby increasing the diagnostic power of the kit. The kit may
contain probes and/or primers that are capable of detecting 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more mutations
in the nucleotide sequence of a human NAGLU gene.
EXAMPLES
[0101] The invention is illustrated by the following example. It is
to be understood that the particular examples, materials, amounts,
and procedures are to be interpreted broadly in accordance with the
scope and spirit of the invention as set forth herein.
Example I. Novel NAGLU Variants Associated with MPS TIM
[0102] In four studies (two natural history, one observational, one
Phase 1/2), we have obtained NAGLU variant data in most patients as
well as dried blood spot testing of NAGLU enzymatic activity. Seven
novel NAGLU variants (c.104T>C, c.351delG, c.1012A>G,
c.1128_1138del11, c.1621C>T, c.1865T>C, IVS2+5G>A) have
been identified in diagnosed patients with MPS IIIB, which may
assist in accurate diagnosis. See Table 1.
[0103] Dried blood spot testing can utilize MPS III/Sanfilippo
enzyme panel, which includes evaluation of
N-acetyl-.alpha.-D-glucosaminidase activity in order to test for
the presence of MPS III, type B. An enzymatic panel can be
obtained, for example, from Greenwood Genetic Center (Greenwood,
S.C.).
Example II. In Vitro Analysis of NAGLU Variants for Residual
Enzymatic Activity
[0104] An in vitro biochemical assay is employed to evaluate the
residual enzymatic activities of NAGLU variants (e.g., those listed
in Table 2). Table 2 includes novel variants described in Example I
and elsewhere herein. All but 52 of the variants were previously
identified in patients. The remaining 52 potentially pathogenic
variants were identified through mining of large population
databases, such as the Exome Aggregation Consortium (ExAC)
database, and are annotated as "predicted" in Table 2.
TABLE-US-00002 TABLE 2 NAGLU Variants Plasmid ID Nucleotide Protein
pNAG012 c.1900G > A E634K pNAG013 c.281_283delinsCCC
R94_D95delinsPH pNAG014 c.1834A > G S612G pNAG015 c.1927C > T
R643C pNAG016 c.529C > T R177W pNAG017 c.1489C > G L497V
pNAG018 c.743A > G H248R pNAG019 c.187G > A D63N pNAG020
c.214-237del24 A72_G79del8 pNAG021 c.1744G > C A582P pNAG022
c.212_214delGCG G71del pNAG023 c.104T > C L35P pNAG024 c.1865T
> C L622P pNAG025 c.1012A > G M338V pNAG026 c.1621C > T
R541W pNAG027 c.1694G > T R565L pNAG028 c.1212G > C W404C
pNAG029 c.507_516del10 5169Rfs*13 pNAG030 c.2186_2188delAGA
K729delK pNAG031 c.813dupT N272*fs*1 pNAG032 c.1128_1138del11
pNAG033 c.351delG pNAG034 c.192delC Y65Tfs*57 pNAG035
c.424_426delTTC F142del pNAG036 c.548G > C G183A predicted
pNAG037 c.593T > A F198Y predicted pNAG038 c.642_643delGCinsGCC
S217LfsX56 predicted pNAG039 c.642_643delGCinsG S217PfsX22
predicted pNAG040 c.727C > T P243S predicted pNAG041 c.730G >
A A244T predicted pNAG042 c.788C > T T263M predicted pNAG043
c.827C > T S276F predicted pNAG044 c.831_835delCTCCTinsC
L280WfsX19 predicted pNAG045 c.842T > C L281P predicted pNAG046
c.848delCinsCG E284GfsX33 predicted pNAG047 c.885delC L296CfsX4
predicted pNAG048 c.917A > G D306G predicted pNAG049 c.979C >
T L327F predicted pNAG050 c.1052G > C G351A predicted pNAG051
c.1061T > C F354S predicted pNAG052 c.1082G > C W361S
predicted pNAG053 c.1120C > T P374S predicted pNAG054
c.1136_1137insG V380GfsX7 predicted pNAG055 c.1154C > T A385V
predicted pNAG056 c.1156G > A E386K predicted pNAG057 c.1196G
> T G399V predicted pNAG058 c.1222C > T H408Y predicted
pNAG059 c.1277G > C G426A predicted pNAG060 c.1291C > T R431C
predicted pNAG061 c.1304A > C N435T predicted pNAG062 c.1360G
> T V454F predicted pNAG063 c.1364A > C Y455S predicted
pNAG064 c.1384G > C G462R predicted pNAG065 c.1390C > T R464X
predicted pNAG066 c.1441C > T R481W predicted pNAG067
c.1446delGinsGT Y483LfsX33 predicted pNAG068 c.1449_1450insG
V485GfsX31 predicted pNAG069 c.1478C > T A493V predicted pNAG070
c.1487T > C L496P predicted pNAG071 c.1495C > T R499W
predicted pNAG072 c.1508A > G N503S predicted pNAG073 c.1538A
> G N513S predicted pNAG074 c.1565C > T S522F predicted
pNAG075 c.1606G > A V536M predicted pNAG076 c.1675G > A D559N
predicted pNAG077 c.1688T > A L563H predicted pNAG078 c.1783G
> A G595R predicted pNAG079 c.1786G > T G596C predicted
pNAG080 c.1918C > T Q640X predicted pNAG081 c.1934A > C Q645P
predicted pNAG082 c.1946G > C W649S predicted pNAG083 c.2017C
> T P673S predicted pNAG085 c.2191T > G F731V predicted
pNAG086 c.2207C > T P736L predicted pNAG087 c.2229G > A W743X
predicted pNAG088 c.100G > C A34P pNAG089 c.1013T > C M338T
pNAG090 c.1354G > A E452K pNAG091 c.1445G > A R482Q pNAG092
c.1444C > T R482W pNAG093 c.14C > T A5V pNAG094 c.1558C >
T R520W pNAG095 c.1547C > G P516R pNAG096 c.1674C > G Y558X
pNAG097 c.1597C > T R533X pNAG098 c.1625T > C L542P pNAG099
c.1675G > C D559H pNAG100 c.1946G > A W649X pNAG101 c.2158C
> T R720X pNAG102 c.2164G > A D722N pNAG103 c.217G > C
A73P pNAG104 c.2209C > A R737S pNAG105 c.2209C > G R737G
pNAG106 c.299A > G H100R pNAG107 c.259G > C A87P pNAG108
c.358G > T E120X pNAG110 c.625A > C T209P pNAG111 c.700C >
G R234G pNAG112 c.814_820dup S274X pNAG113 c.934G > A D312N
pNAG114 c.1039_1040delTG W347Afs*39 pNAG115 c.1691_1694dupCTCG
Q566Sfs*13 pNAG116 c.171_174dupTGCC K59Cfs*134 pNAG117
c.1815_1821dupACTGGAC E608Tfs*7 pNAG118 c.1928_1932dupGCTAC
Q645Afs*4 pNAG119 c.193delT Y65Tfs*57 pNAG120 c.1951_1954dupCCAG
E652Afs*34 pNAG121 c.1004A > G Y335C pNAG122 c.1006G > T
E336X pNAG123 c.103C > T L35F pNAG124 c.1000G > T V334F
pNAG125 c.112C > T R38W pNAG126 c.1229T > C F410S pNAG127
c.1235G > A G412E pNAG128 c.1241A > G H414R pNAG129 c.1310C
> T T437I pNAG130 c.1322C > A T441K pNAG131 c.1336G > A
E446K pNAG132 c.1420T > G W474G pNAG133 c.144C > A F48L
pNAG134 c.1482G > A W494X pNAG135 c.1547C > T P516L pNAG136
c.1502T > G V501G pNAG137 c.1601C > A S534Y pNAG138 c.1682T
> G L561R pNAG139 c.1693C > T R565W pNAG140 c.1694G > A
R565Q pNAG141 c.1811C > T P604L pNAG142 c.1831G > C A611P
pNAG143 c.1851G > C L617F pNAG144 c.1915G > T E639X pNAG145
c.1946G > T W649L pNAG146 c.1947G > C W649C pNAG147 c.1949G
> A G650E pNAG148 c.1973A > T Y658F pNAG149 c.2021G > A
R674H pNAG150 c.205G > A G69S pNAG151 c.208G > C G70R pNAG152
c.220dupC R74Pfs*118 pNAG153 c.38_39insC L14Sfs*178 pNAG154
c.410_413delCGCA T137Kfs*17 pNAG155 c.507_515delCGGCCAGGA
S169_E172delinsR pNAG156 c.54_60dupCGGGGGC A21Rfs*173 pNAG157
c.651dupC W218Lfs*55 pNAG158 c.902_903delAA K301Rfs*15 pNAG159
c.905_906delAG E302Vfs*14 pNAG160 c.950_951insAA M317Ifs*23 pNAG161
c.230T > G V77G pNAG162 c.245G > A G82D pNAG163 c.388C > T
R130C pNAG164 c.422C > T S141F pNAG165 c.441G > A W147X
pNAG166 c.461T > G I154R pNAG167 c.468G > T W156C pNAG168
c.482G > A G161D pNAG169 c.680A > C H227P pNAG170 c.721G >
A V241M pNAG171 c.725T > C L242P pNAG172 c.728C > T P243L
pNAG173 c.830G > T C277F pNAG174 c.839T > C L280P pNAG175
c.926A > G Y309C pNAG176 c.940T > C F314L pNAG177 c.142T >
C F48L pNAG178 c.235G > A G79S pNAG179 c.1679T > C L560P
pNAG180 c.736G > C A246P pNAG181 c.1694G > C R565P pNAG182
c.630G > C W210C pNAG183 c.334delC R112Gfs*10 pNAG184 c.504delG
W168*fs*1 pNAG185 c.233_ R78_ 234ins G79ins GCGGCGCGGCGCGC RRGARAGA
GTGCGGGTGC pNAG186 c.814_816delAACinsTAA N272X pNAG187
c.222_247del26 V75Gfs*108 pNAG188 c.903delA E302Sfs*37 pNAG189
c.241A > G T81A pNAG190 c.392A > C Y131S pNAG191 c.410C >
T T137M pNAG192 c.432G > A W144X pNAG193 c.461T > C I154T
pNAG194 c.472G > T A158S pNAG195 c.845C > T A282V pNAG196
c.1991C > T A664V pNAG197 c.214-237dup24 A72_G79dup8 pNAG198
c.457G > A E153K pNAG199 c.2113G > A E705K pNAG200 c.942C
> G F314L pNAG201 c.143T > G F48C pNAG202 c.874G > A G292R
pNAG203 c.911G > T G304V pNAG204 c.235G > T G79C pNAG205
c.1208T > C I403T pNAG206 c.1772T > C L591P pNAG207 c.200T
> C L67P pNAG208 c.2045T > G L682R pNAG209 c.343C > T
P115S pNAG210 c.1073C > T P358L pNAG211 c.1562C > T P521L
pNAG212 c.2116C > T Q706X pNAG213 c.607C > T R203X pNAG214
c.700C > T R234C pNAG215 c.889C > T R297X pNAG216 c.1876C
> T R626X pNAG217 c.1928G > A R643H pNAG218 c.2020C > T
R674C pNAG219 c.2027G > C R676P pNAG220 c.1564T > C S522P
pNAG221 c.217_221dupGCGCG V75Rfs*49 pNAG222 c.802T > C W268R
pNAG223 c.1081T > C W361R pNAG224 c.1211G > A W404X pNAG225
c.2024G > A W675X pNAG226 c.419A > G Y140C pNAG227 c.1172A
> G Y391C pNAG228 c.1364A > G Y455C pNAG229 c.274T > C
Y92H pNAG230 c.1487delT L496Hfs*30 pNAG231 c.204delC G69Afs*53
pNAG232 c.407_410del4 pNAG233 c.507delC S169Rfs*16 pNAG234
c.703delT S235Pfs*4 pNAG235 c.1317delA G440Afs*36 pNAG236
c.1335delC E446Rfs*30 pNAG237 c.1447dupT Y483Lfs*33 pNAG238
c.1932-1933insGCTAC pNAG241 c.219_234del19 R74Pfs*42 pNAG242
c.334_358del25 R112Sfs*2 pNAG243 c.59delG G20Afs*102 pNAG244
c.867delC I290Sfs*10 pNAG246 c.503G > A W168X pNAG247 c.660delC
K221Sfs*18
Expression of NAGLU Variants
[0105] Plasmids encoding wild-type (WT) NAGLU and NAGLU variants
with C-terminal 6.times. histidine tags were ordered from Thermo
Fisher Scientific GeneArt (Regensburg, Germany). Plasmids were
sequence verified to confirm the presence of desired mutations.
Constructs were transiently transfected into Expi293F cells using
ExpiFectamine 293 and the methodology recommended by the
manufacturer (Thermo Fisher Scientific, Carlsbad, Calif.).
Transfections were carried out at the two milliliter scale in
12-well tissue culture plates (Fisher Scientific, Waltham, Mass.).
Transfected cultures were harvested three days post-transfection.
Briefly, cultures were spun down at 500.times.g for five minutes,
supernatants transferred to fresh plates, and cell pellets washed
twice in phosphate-buffered saline (PBS, GE Healthcare,
Marlborough, Mass.). Transfected cultures were incubated with 0.5
mL lysis buffer [1% Triton X-100, 10 mM Sodium Phosphate (pH 7.0),
10 mM dithiothreitol (DTT) and 1 mM ethylenediaminetetraacetic acid
(EDTA) in water] for 45 minutes at 4.degree. C. and centrifuged for
15 minutes at 3,000.times.g to remove insoluble materials.
Western Blot
[0106] Cell lysates and supernatants from transfected cultures were
mixed with 4.times.E-PAGE loading buffer and run on 48-well E-PAGE
8% Protein Gels (Thermo Fisher Scientific). Proteins were
transferred to polyvinylidene fluoride (PVDF) membranes (Thermo
Fisher Scientific), which were incubated overnight in blocking
buffer [1% bovine serum albumin (BSA), 0.05% Tween-20 (both
Sigma-Aldrich, St. Louis, Mo.) in PBS] at 4.degree. C. Membranes
were probed with 1 .mu.g/mL of mouse monoclonal anti-6.times. his
tag antibody (Abcam, Cambridge, Mass.) in wash buffer [0.3% BSA,
0.05% Tween-20 in PBS] for 60 minutes, washed five times, incubated
with 0.1 .mu.g/mL IRDye 680RD Donkey anti-mouse IgG (H+L) (LiCor,
Lincoln, Nebr.) for thirty minutes and washed four times in wash
buffer and once in PBS. His-tagged proteins were detected by
near-infrared fluorescence of the secondary antibody using the
Odyssey CLx (LiCor).
NAGLU Enzyme Assay
[0107] Cell lysate or supernatant from transfected cells (40 .mu.l)
was transferred to a black 384 well Optiplate (Perkin Elmer,
Waltham, Mass.). The NAGLU enzyme reaction was started by adding 20
.mu.l of the substrate
4-methylumbelliferyl-N-acetyl-.alpha.-D-glucosaminide (EMD
Millipore/SigmaAldrich) in 1.times. assay buffer [100 mM Sodium
Acetate (pH 5.5) and 250 mM NACl] at a final concentration of 375
.mu.M. 4-methylumbelliferyl N-acetyl-.alpha.-D-glucosaminide is
also known as 4-methylumbelliferyl
2-acetamido-2-deoxy-.alpha.-D-glucopyranoside, (MUG), and is
available from Santa Cruz Biotechnology as well. The final reaction
volume was 60 .mu.l. A BioTek Synergy 2 plate reader was used to
follow 4-methylumbelliferyl fluorophore production at
excitation/emission wavelengths of 360/460+/-40 nm. The initial
velocity for each NAGLU variant was determined from the first 1 to
2 hours of the reaction. Total protein for each variant was
determined (Pierce BCA protein assay kit) and activity was
normalized to WT protein levels. Variant NAGLU activity was then
compared to WT NAGLU activity.
Example III. Variant Analysis and In Silico Prediction of Mutation
Severity
Methods
[0108] Allelic variants of NAGLU in patients enrolled in 3 clinical
studies (2 natural history and 1 phase I/II) of the SBC-103
(rhNAGLU enzyme) clinical program were analyzed. NGLU-NH01 is a
natural history study of deceased MPS TIM patients. Historical
mutational data from local laboratories was available from 8 of the
30 patients included in the retrospective study. NGLU-NH0 2 is a
prospective natural history study of MPS IIIB patients (30
patients). NGLU-CLO2 is an ongoing phase I/II investigational study
of SBC-103 in MPS IIIB patients (11 patients).
[0109] In NGLU-NH02 and NGLU-CLO2 studies, a blood sample for DNA
extraction was collected from each subject and isolation of DNA and
genetic mutation analysis for allelic variants of the NAGLU gene
was performed.
[0110] Four in silico prediction methods (MutationTaster,
PolyPhen2, SIFT, and Provean) were utilized to assess the severity
of missense mutations and to predict the impact of the mutation on
NAGLU enzymatic activity. Missense mutations were flagged based on
a consensus score of the four methods. A mutation was flagged as
"deleterious" if all four methods predicted it as "deleterious,"
otherwise it was flagged as "benign."
Results
[0111] A total of 53 unique NAGLU variants were identified in 49
patients whose genetic data were available. Of the 53 mutations, 45
variants were identified previously and 8 are novel variants (Table
3). Example II shows 7 of the 8 novel variants; this study
identified an additional novel variant, namely, c.1700_1708del9,
resulting in an in frame deletion, A567_Q569del.
TABLE-US-00003 TABLE 3 Novel NAGLU Variants Identified in Patients
Variant Domain(s) In silico Nucleotide type Amino acid affected
prediction c.1012A > G missense M338V TIM barrel deleterious
c.1621C > T missense R541W C-terminal benign c.1865T > C
missense L622P C-terminal deleterious IVS2 + 5G > A.sup.a splice
TIM barrel, variant C-terminal c.104T > C missense L35P before
deleterious N-terminal c.1128_1138del11.sup.a frameshift TIM
barrel, C-terminal c.351delG.sup.a frameshift TIM barrel,
C-terminal c.1700_1708del9 in frame A567_Q569del C-terminal
deletion .sup.aFor frameshift and spice variants, the affected
domain(s) theoretically starts where the variant is, and extends to
the end of the protein.
[0112] No highly enriched variant was observed: the majority of the
variants (39), including all novel variants, were present only once
in this patient group, whereas the other 14 variants were present
multiple times with c.419A>G as the most frequent allele at 5
times (Table 4).
TABLE-US-00004 TABLE 4 NAGLU Variants That Were Identified More
Than One Time in Patients Amino Domain(s) Nucleotide Variant type
acid affected Frequency c.419A > G missense Y140C TIM barrel 5
c.700C > T missense R234C TIM barrel 4 c.1211G > A nonsense
W404X TIM barrel 4 c.874G > A missense G292R TIM barrel 4 c.889C
> T nonsense R297X TIM barrel, 3 C-terminal c.1694G > A
missense R565Q C-terminal 2 c.1562C > T missense P521L
C-terminal 2 c.503G > A nonsense W168X TIM barrel, 2 C-terminal
c.507_516del10 frameshift TIM barrel, 2 C-terminal c.1834A > G
missense S612G C-terminal 2 c.2186_ in-frame K729del C-terminal end
2 2188delAGA deletion c.889C > T nonsense R297X TIM barrel, 2
C-terminal c.212_ in-frame G71del N-terminal 2 214delGCG deletion
c.192delC frameshift N-terminal, TIM 2 barrel, C-terminal
[0113] NAGLU has three conserved domains: an N-terminal domain
spanning amino acids 42-116; a TIM barrel domain spanning amino
acids 130-465; and a C-terminal domain spanning amino acids
474-729. As shown in FIG. 3, all but one (c.104T>C) of the 53
NAGLU variant mutations are located in one of the three conserved
domains in the NAGLU enzyme. Mutations may affect one or more
domains of the protein. About half of the mutations are present in
the TIM barrel domain and another half in the C-terminal domain; 6
mutations affect only the N-terminal domain.
[0114] The complete disclosures of all patents, patent applications
including provisional patent applications, publications including
patent publications and nonpatent publications, and electronically
available material (including, for example, nucleotide sequence
submissions in, e.g., GenBank and RefSeq, and amino acid sequence
submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations
from annotated coding regions in GenBank and RefSeq) cited herein
are incorporated by reference.
[0115] The foregoing detailed description and examples have been
given for clarity of understanding only. No unnecessary limitations
are to be understood therefrom. The invention is not limited to the
exact details shown and described; many variations will be apparent
to one skilled in the art and are intended to be included within
the invention defined by the claims.
Sequence CWU 1
1
31743PRThomo sapien 1Met Glu Ala Val Ala Val Ala Ala Ala Val Gly
Val Leu Leu Leu Ala 1 5 10 15 Gly Ala Gly Gly Ala Ala Gly Asp Glu
Ala Arg Glu Ala Ala Ala Val 20 25 30 Arg Ala Leu Val Ala Arg Leu
Leu Gly Pro Gly Pro Ala Ala Asp Phe 35 40 45 Ser Val Ser Val Glu
Arg Ala Leu Ala Ala Lys Pro Gly Leu Asp Thr 50 55 60 Tyr Ser Leu
Gly Gly Gly Gly Ala Ala Arg Val Arg Val Arg Gly Ser 65 70 75 80 Thr
Gly Val Ala Ala Ala Ala Gly Leu His Arg Tyr Leu Arg Asp Phe 85 90
95 Cys Gly Cys His Val Ala Trp Ser Gly Ser Gln Leu Arg Leu Pro Arg
100 105 110 Pro Leu Pro Ala Val Pro Gly Glu Leu Thr Glu Ala Thr Pro
Asn Arg 115 120 125 Tyr Arg Tyr Tyr Gln Asn Val Cys Thr Gln Ser Tyr
Ser Phe Val Trp 130 135 140 Trp Asp Trp Ala Arg Trp Glu Arg Glu Ile
Asp Trp Met Ala Leu Asn 145 150 155 160 Gly Ile Asn Leu Ala Leu Ala
Trp Ser Gly Gln Glu Ala Ile Trp Gln 165 170 175 Arg Val Tyr Leu Ala
Leu Gly Leu Thr Gln Ala Glu Ile Asn Glu Phe 180 185 190 Phe Thr Gly
Pro Ala Phe Leu Ala Trp Gly Arg Met Gly Asn Leu His 195 200 205 Thr
Trp Asp Gly Pro Leu Pro Pro Ser Trp His Ile Lys Gln Leu Tyr 210 215
220 Leu Gln His Arg Val Leu Asp Gln Met Arg Ser Phe Gly Met Thr Pro
225 230 235 240 Val Leu Pro Ala Phe Ala Gly His Val Pro Glu Ala Val
Thr Arg Val 245 250 255 Phe Pro Gln Val Asn Val Thr Lys Met Gly Ser
Trp Gly His Phe Asn 260 265 270 Cys Ser Tyr Ser Cys Ser Phe Leu Leu
Ala Pro Glu Asp Pro Ile Phe 275 280 285 Pro Ile Ile Gly Ser Leu Phe
Leu Arg Glu Leu Ile Lys Glu Phe Gly 290 295 300 Thr Asp His Ile Tyr
Gly Ala Asp Thr Phe Asn Glu Met Gln Pro Pro 305 310 315 320 Ser Ser
Glu Pro Ser Tyr Leu Ala Ala Ala Thr Thr Ala Val Tyr Glu 325 330 335
Ala Met Thr Ala Val Asp Thr Glu Ala Val Trp Leu Leu Gln Gly Trp 340
345 350 Leu Phe Gln His Gln Pro Gln Phe Trp Gly Pro Ala Gln Ile Arg
Ala 355 360 365 Val Leu Gly Ala Val Pro Arg Gly Arg Leu Leu Val Leu
Asp Leu Phe 370 375 380 Ala Glu Ser Gln Pro Val Tyr Thr Arg Thr Ala
Ser Phe Gln Gly Gln 385 390 395 400 Pro Phe Ile Trp Cys Met Leu His
Asn Phe Gly Gly Asn His Gly Leu 405 410 415 Phe Gly Ala Leu Glu Ala
Val Asn Gly Gly Pro Glu Ala Ala Arg Leu 420 425 430 Phe Pro Asn Ser
Thr Met Val Gly Thr Gly Met Ala Pro Glu Gly Ile 435 440 445 Ser Gln
Asn Glu Val Val Tyr Ser Leu Met Ala Glu Leu Gly Trp Arg 450 455 460
Lys Asp Pro Val Pro Asp Leu Ala Ala Trp Val Thr Ser Phe Ala Ala 465
470 475 480 Arg Arg Tyr Gly Val Ser His Pro Asp Ala Gly Ala Ala Trp
Arg Leu 485 490 495 Leu Leu Arg Ser Val Tyr Asn Cys Ser Gly Glu Ala
Cys Arg Gly His 500 505 510 Asn Arg Ser Pro Leu Val Arg Arg Pro Ser
Leu Gln Met Asn Thr Ser 515 520 525 Ile Trp Tyr Asn Arg Ser Asp Val
Phe Glu Ala Trp Arg Leu Leu Leu 530 535 540 Thr Ser Ala Pro Ser Leu
Ala Thr Ser Pro Ala Phe Arg Tyr Asp Leu 545 550 555 560 Leu Asp Leu
Thr Arg Gln Ala Val Gln Glu Leu Val Ser Leu Tyr Tyr 565 570 575 Glu
Glu Ala Arg Ser Ala Tyr Leu Ser Lys Glu Leu Ala Ser Leu Leu 580 585
590 Arg Ala Gly Gly Val Leu Ala Tyr Glu Leu Leu Pro Ala Leu Asp Glu
595 600 605 Val Leu Ala Ser Asp Ser Arg Phe Leu Leu Gly Ser Trp Leu
Glu Gln 610 615 620 Ala Arg Ala Ala Ala Val Ser Glu Ala Glu Ala Asp
Phe Tyr Glu Gln 625 630 635 640 Asn Ser Arg Tyr Gln Leu Thr Leu Trp
Gly Pro Glu Gly Asn Ile Leu 645 650 655 Asp Tyr Ala Asn Lys Gln Leu
Ala Gly Leu Val Ala Asn Tyr Tyr Thr 660 665 670 Pro Arg Trp Arg Leu
Phe Leu Glu Ala Leu Val Asp Ser Val Ala Gln 675 680 685 Gly Ile Pro
Phe Gln Gln His Gln Phe Asp Lys Asn Val Phe Gln Leu 690 695 700 Glu
Gln Ala Phe Val Leu Ser Lys Gln Arg Tyr Pro Ser Gln Pro Arg 705 710
715 720 Gly Asp Thr Val Asp Leu Ala Lys Lys Ile Phe Leu Lys Tyr Tyr
Pro 725 730 735 Arg Trp Val Ala Gly Ser Trp 740 22232DNAhomo sapien
2atggaggcgg tggcggtggc cgcggcggtg ggggtccttc tcctggccgg ggccgggggc
60gcggcaggcg acgaggcccg ggaggcggcg gccgtgcggg cgctcgtggc ccggctgctg
120gggccaggcc ccgcggccga cttctccgtg tcggtggagc gcgctctggc
tgccaagccg 180ggcttggaca cctacagcct gggcggcggc ggcgcggcgc
gcgtgcgggt gcgcggctcc 240acgggcgtgg cggccgccgc ggggctgcac
cgctacctgc gcgacttctg tggctgccac 300gtggcctggt ccggctctca
gctgcgcctg ccgcggccac tgccagccgt gccgggggag 360ctgaccgagg
ccacgcccaa caggtaccgc tattaccaga atgtgtgcac gcaaagctac
420tctttcgtgt ggtgggactg ggcccgctgg gagcgagaga tagactggat
ggcgctgaat 480ggcatcaacc tggcactggc ctggagcggc caggaggcca
tctggcagcg ggtgtacctg 540gccttgggcc tgacccaggc agagatcaat
gagttcttta ctggtcctgc cttcctggcc 600tgggggcgaa tgggcaacct
gcacacctgg gatggccccc tgcccccctc ctggcacatc 660aagcagcttt
acctgcagca ccgggtcctg gaccagatgc gctccttcgg catgacccca
720gtgctgcctg cattcgcggg gcatgttccc gaggctgtca ccagggtgtt
ccctcaggtc 780aatgtcacga agatgggcag ttggggccac tttaactgtt
cctactcctg ctccttcctt 840ctggctccgg aagaccccat attccccatc
atcgggagcc tcttcctgcg agagctgatc 900aaagagtttg gcacagacca
catctatggg gccgacactt tcaatgagat gcagccacct 960tcctcagagc
cctcctacct tgccgcagcc accactgccg tctatgaggc catgactgca
1020gtggatactg aggctgtgtg gctgctccaa ggctggctct tccagcacca
gccgcagttc 1080tgggggcccg cccagatcag ggctgtgctg ggagctgtgc
cccgtggccg cctcctggtt 1140ctggacctgt ttgctgagag ccagcctgtg
tatacccgca ctgcctcctt ccagggccag 1200cccttcatct ggtgcatgct
gcacaacttt gggggaaacc atggtctttt tggagcccta 1260gaggctgtga
acggaggccc agaagctgcc cgcctcttcc ccaactccac catggtaggc
1320acgggcatgg cccccgaggg catcagccag aacgaagtgg tctattccct
catggctgag 1380ctgggctggc gaaaggaccc agtgccagat ttggcagcct
gggtgaccag ctttgccgcc 1440cggcggtatg gggtctccca cccggacgca
ggggcagcgt ggaggctact gctccggagt 1500gtgtacaact gctccgggga
ggcctgcagg ggccacaatc gtagcccgct ggtcaggcgg 1560ccgtccctac
agatgaatac cagcatctgg tacaaccgat ctgatgtgtt tgaggcctgg
1620cggctgctgc tcacatctgc tccctccctg gccaccagcc ccgccttccg
ctacgacctg 1680ctggacctca ctcggcaggc agtgcaggag ctggtcagct
tgtactatga ggaggcaaga 1740agcgcctacc tgagcaagga gctggcctcc
ctgttgaggg ctggaggcgt cctggcctat 1800gagctgctgc cggcactgga
cgaggtgctg gctagtgaca gccgcttctt gctgggcagc 1860tggctagagc
aggcccgagc agcggcagtc agtgaggccg aggccgattt ctacgagcag
1920aacagccgct accagctgac cttgtggggg ccagaaggca acatcctgga
ctatgccaac 1980aagcagctgg cggggttggt ggccaactac tacacccctc
gctggcggct tttcctggag 2040gcgctggttg acagtgtggc ccagggcatc
cctttccaac agcaccagtt tgacaaaaat 2100gtcttccaac tggagcaggc
cttcgttctc agcaagcaga ggtaccccag ccagccgcga 2160ggagacactg
tggacctggc caagaagatc ttcctcaaat attacccccg ctgggtggcc
2220ggctcttggt ga 2232323PRThomo sapien 3Met Glu Ala Val Ala Val
Ala Ala Ala Val Gly Val Leu Leu Leu Ala 1 5 10 15 Gly Ala Gly Gly
Ala Ala Gly 20
* * * * *
References